This invention relates to a method for vitrification of a biological specimen, such that the biological specimen remains viable after it is thawed.
The ability to cryopreserve oocytes, embryos, sperm and other similar biological specimens is critical to the widespread application of assisted reproductive technologies. However, due to the large volume of the cells and the high chilling sensitivity of oocytes and early embryos, cryopreservation techniques are not well developed in most species.
Traditionally, embryos are cryopreserved using xe2x80x9cslow freezing techniquesxe2x80x9d. Low concentrations of cryoprotectants and slow controlled rates of cooling usually in the range of 0.1-10.3xc2x0 C./min. slowly dehydrate the cell during freezing to prevent intracellular crystallization. Because of this, cryopreservation of oocytes, embryos and other developmental cells using such procedures results in a reduced ability to both establish and maintain pregnancy following transfer. Oocytes are particularly susceptible to cryopreservation damage because of disruption of the metaphase spindle microtubule integrity during cooling.
Alternative prior cryopreservation methods have relied on vitrification with high concentrations of cryoprotectants, which when rapidly cooled result in a glass-like state. However, a disadvantage of this vitrification technique is that the cryoprotectants are very toxic to oocytes, embryos and other delicate developmental cells. Cryoprotectant toxicity can be minimized by increasing the cooling rate, which has been accomplished by plunging oocytes held on electron microscopy grids, or within thinly walled straws (known as open pulled straw) directly into liquid nitrogen. However, both of these procedures are cumbersome and recovery of embryos is problematic.
Therefore a need remains for a method for the vitrification of a biological specimen which is able to maximize the cooling rate of the cells of the specimen; maintain viability of the specimen during vitrification and subsequent thawing; prevent mechanical stress to the specimen; and provide ease of manipulations during cryopreservation and recovery.
The present invention relates to a method of vitrification of a biological specimen. According to the method of the present invention, a biological specimen is directly exposed to a freezing material. Upon exposure to the freezing material, the biological specimen undergoes vitrification. The biological specimen which has undergone vitrification may be stored for a period of time, and then thawed at a later date. The thawed biological specimen remains viable. Preferred biological specimens according to the present invention are developmental cells.
The present invention is also drawn to a method of vitrification of a biological specimen, which includes using a transfer instrument to place the biological specimen into a freezing material, such as liquid nitrogen, such that the biological specimen is directly exposed to the freezing material. The biological specimen then undergoes vitrification while held by the transfer instrument, with a loop being a preferred transfer instrument. The transfer instrument and biological specimen are then preferably kept within the freezing material, and transferred into a container which holds a freezing material. The container is preferably a vial. The vial is then sealed containing the freezing material, loop and the vitrified biological specimen, and may be cryopreserved until such time as the biological specimen is required for further use.
Another aspect of the present invention is the treatment of the biological specimen in a cryoprotectant prior to vitrification.
The invention also relates to a method for thawing a biological specimen which has undergone vitrification. The thawing methodology comprises the removal of the biological specimen from the freezing material wherein it has been cryopreserved, and placing the biological specimen in a warmed thaw solution. The thaw solution may be present in any suitable container, and is preferably located within a culture dish or a straw.
A further aspect of the present invention is a method of vitrification of developmental cells, wherein one or more developmental cells are placed directly into a freezing material, such that each developmental cell is directly exposed to the freezing material thereby undergoing vitrification, wherein the vitrified developmental cells, when thawed, cultured and implanted into suitable host organisms, will result in a fertility rate equal to that of the same developmental cells which had not been vitrified. Preferably, the developmental cells are contained within a loop when exposed to the freezing material.
The present invention also relates to a method of vitrification of a mammalian blastocyst or mammalian cleavage stage embryo which comprises placing one or more blastocysts or cleave stage embryos directly into a freezing material, such that each blastocyst or cleavage stage embryo is directly exposed to the freezing material thereby undergoing vitrification, wherein at least 80 percent, and more preferably, 90 percent, of the vitrified blastocysts or cleavage stage embryos will be viable after being thawed and cultured, preferably in the appropriate base medium. Preferably, the blastocyst or cleavage stage embryo is contained within a loop when exposed to the freezing material.
The present invention also relates to a method of vitrification of a horse embryo or pig embryo which comprises placing one or more embryos directly into a freezing material, such that each embryo is directly exposed to the freezing material thereby undergoing vitrification, wherein at least 25 percent, and more preferably, 50 percent, of the vitrified embryos will be viable after being thawed and cultured, preferably in the appropriate base medium. Preferably, the embryo is contained within a loop when exposed to the freezing material.
The present invention also relates to a kit for the vitrification of a biological specimen. The kit will generally contain instructions describing the vitrification of a biological specimen wherein the specimen is directly exposed to a freezing material. The kit will also include one or more optional ingredients, including, but not limited to, a transfer instrument, most preferably a loop, a vial which is of the proper size and shape to hold the loop and the vitrified specimen it contains, a base medium, a transfer solution, and a cryoprotectant.
The present invention is also drawn to biological specimens which have undergone vitrification by the methods of the present invention.